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Serum-derived bovine immunoglobulin (SBI) prevents translocation and inflammation via direct binding of microbial components. Recently, SBI also displayed potential benefits through gut microbiome modulation. To confirm and expand upon these preliminary findings, SBI digestion and colonic fermentation were investigated using the clinically predictive ex vivo SIFR ® technology (for 24 human adults) that was, for the first time, combined with host cells (epithelial/immune (Caco-2/THP-1) cells). SBI (human equivalent dose (HED) = 2 and 5 g/day) and the reference prebiotic inulin (IN; HED = 2 g/day) significantly promoted gut barrier integrity and did so more profoundly than a dietary protein (DP), especially upon LPS-induced inflammation. SBI also specifically lowered inflammatory markers (TNF-α and CXCL10). SBI and IN both enhanced SCFA (acetate/propionate/butyrate) via specific gut microbes, while SBI specifically stimulated valerate/bCFA and indole-3-propionic acid (health-promoting tryptophan metabolite). Finally, owing to the high-powered cohort (n = 24), treatment effects could be stratified based on initial microbiota composition: IN exclusively stimulated (acetate/non-gas producing) Bifidobacteriaceae for subjects classifying as Bacteroides / Firmicutes -enterotype donors, coinciding with high acetate/low gas production and thus likely better tolerability of IN. Altogether, this study strongly suggests gut microbiome modulation as a mechanism by which SBI promotes health. Moreover, the SIFR ® technology was shown to be a powerful tool to stratify treatment responses and support future personalized nutrition approaches.

The human gut microbiota is characterized by large interpersonal differences, which are not only linked to health and disease but also determine the outcome of nutritional interventions. In line with the growing interest for developing targeted gut microbiota modulators, the selectivity of a carrot-derived rhamnogalacturonan I (cRG-I) was compared to substrates with demonstrated low (inulin, IN) and high selectivity (xanthan, XA), at a human equivalent dose (HED) of 1.5 g/d. The high throughput of the ex vivo SIFR ® technology, validated to generate predictive insights for clinical findings, enabled the inclusion of 24 human adults. Such an unprecedented high number of samples in the context of in vitro gut microbiota modelling allowed a coverage of clinically relevant interpersonal differences in gut microbiota composition and function. A key finding was that cRG-I supplementation (already at an HED of 0.3 g/d) lowered interpersonal compositional differences due to the selective stimulation of taxa that were consistently present among human adults, including OTUs related to Bacteroides dorei/vulgatus and Bifidobacterium longum (suspected keystone species) , Bacteroides thetaiotaomicron , Bifidobacterium adolescentis and butyrate-producing taxa such as Blautia sp., Anaerobutyricum hallii, and Faecalibacterium prausnitzii . In contrast, both IN and XA treatments increased interpersonal compositional differences. For IN, this followed from its low specificity. For XA, it was rather the extremely high selectivity of XA fermentation that caused large differences between 15 responders and 9 nonresponders, caused by the presence/absence of highly specific XA-fermenting taxa. While all test compounds significantly enhanced acetate, propionate, butyrate, and gas production, cRG-I resulted in a significantly higher acetate (+40%), propionate (+22%), yet a lower gas production (-44%) compared to IN. cRG-I could thus result in overall more robust beneficial effects, while also being better tolerated. Moreover, owing to its remarkable homogenization effect on microbial composition and metabolite production, cRG-I could lead to more predictable outcomes compared to substrates that are less specific or overly specific.

Prebiotics are substrates that are selectively utilized by host microorganisms, thus conferring a health benefit. There is a growing awareness that interpersonal and age-dependent differences in gut microbiota composition impact prebiotic effects. Due to the interest in using human milk oligosaccharides (HMOs) beyond infancy, this study evaluated how HMOs [2'Fucosyllactose (2'FL), Lacto-N-neotetraose (LNnT), 3'Sialyllactose (3'SL), 6'Sialyllactose (6'SL)] and blends thereof affect the microbiota of 6-year-old children ( n = 6) and adults ( n = 6), compared to prebiotics inulin (IN) and fructooligosaccharides (FOS). The ex vivo SIFR ® technology was used, given its demonstrated predictivity in clinical findings. First, HMOs and HMO blends seemed to maintain a higher α-diversity compared to FOS/IN. Further, while 2'FL/LNnT were bifidogenic for both age groups, 3'SL/6'SL and FOS/IN were exclusively bifidogenic for children and adults, respectively. This originated from age-related differences in microbiota composition because while 3'SL/6'SL stimulated B. pseudocatenulatum (abundant in children), FOS/IN enhanced B. adolescentis (abundant in adults). Moreover, all treatments significantly increased acetate, propionate and butyrate (only in adults) with product- and age-dependent differences. Among the HMOs, 6'SL specifically stimulated propionate (linked to Bacteroides fragilis in children and Phocaeicola massiliensis in adults), while LNnT stimulated butyrate (linked to Anaerobutyricum hallii in adults). Indole-3-lactic acid and 3-phenyllactic acid (linked to immune health) and gamma-aminobutyric acid (linked to gut-brain axis) were most profoundly stimulated by 2'FL and HMO blends in both children and adults, correlating with specific Bifidobacteriaceae . Finally, 2'FL/LNnT increased melatonin in children, while 3'SL remarkably increased folic acid in adults. Overall, age-dependent differences in microbiota composition greatly impacted prebiotic outcomes, advocating for the development of age-specific nutritional supplements. HMOs were shown to be promising modulators in the adult, and particularly the children's microbiota. The observed HMO-specific effects, likely originating from their structural heterogeneity, suggest that blends of different HMOs could maximize treatment effects.

Background: The infant gut microbiota establishes during a critical window of opportunity when metabolic and immune functions are highly susceptible to environmental changes, such as diet. Human milk oligosaccharides (HMOs) for instance are suggested to be beneficial for infant health and gut microbiota. Infant formulas supplemented with the HMOs 2'-fucosyllactose (2'-FL) and lacto-N-neotetraose (LNnT) reduce infant morbidity and medication use and promote beneficial bacteria in the infant gut ecosystem. To further improve infant formula and achieve closer proximity to human milk composition, more complex HMO mixtures could be added. However, we currently lack knowledge about their effects on infants' gut ecosystems., Method: We assessed the effect of lactose, 2'-FL, 2'-FL + LNnT, and a mixture of six HMOs (HMO6: consisting of 2'-FL, LNnT, difucosyllactose, lacto-N-tetraose, 3'- and 6'-sialyllactose) on infant gut microbiota and intestinal barrier integrity using a combination of in vitro models to mimic the microbial ecosystem (baby M-SHIME ® ) and the intestinal epithelium (Caco-2/HT29-MTX co-culture)., Results: All the tested products had bifidogenic potential and increased SCFA levels; however, only the HMOs' fermented media protected against inflammatory intestinal barrier disruption. 2'-FL/LNnT and HMO6 promoted the highest diversification of OTUs within the Bifidobactericeae family, whereas beneficial butyrate-producers were specifically enriched by HMO6., Conclusion: These results suggest that increased complexity in HMO mixture composition may benefit the infant gut ecosystem, promoting different bifidobacterial communities and protecting the gut barrier against pro-inflammatory imbalances.

The human gut microbiota has been linked to the health status of the host. Modulation of human gut microbiota through pro- and prebiotic interventions has yielded promising results; however, the effect of novel prebiotics, such as chitin-glucan, on gut microbiota-host interplay is still not fully characterized. We assessed the effect of chitin-glucan (CG) and chitin-glucan plus Bifidobacterium breve (CGB) on human gut microbiota from the luminal and mucosal environments in vitro. Further, we tested the effect of filter-sterilized fecal supernatants from CG and CGB fermentation for protective effects on inflammation-induced barrier disruption and cytokine production using a co-culture of enterocytes and macrophage-like cells. Overall, CG and CGB promote health-beneficial short-chain fatty acid production and shift human gut microbiota composition, with a consistent effect increasing Roseburia spp. and butyrate producing-bacteria. In two of three donors, CG and CGB also stimulated Faecalibacterium prausniitzi . Specific colonization of B. breve was observed in the lumen and mucosal compartment; however, no synergy was detected for different endpoints when comparing CGB and CG. Both treatments included a significant improvement of inflammation-disrupted epithelial barrier and shifts on cytokine production, especially by consistent increase in the immunomodulatory cytokines IL10 and IL6.

Human gut microbiota (HGM) play a significant role in health and disease. Dietary components, including fiber, fat, proteins and micronutrients, can modulate HGM. Much research has been performed on conventional prebiotics such as fructooligosaccharides (FOS) and galactooligosaccharides (GOS), however, novel prebiotics or micronutrients still require further validation. We assessed the effect of FOS, xylooligosaccharides (XOS) and a mixture of an antioxidant vitamin blend (AOB) on gut microbiota composition and activity, and intestinal barrier in vitro. We used batch fermentations and tested the short-term effect of different products on microbial activity in six donors. Next, fecal inocula from two donors were used to inoculate the simulator of the human microbial ecosystem (SHIME) and after long-term exposure of FOS, XOS and AOB, microbial activity (short- and branched-chain fatty acids and lactate) and HGM composition were evaluated. Finally, in vitro assessment of intestinal barrier was performed in a Transwell setup of differentiated Caco-2 and HT29-MTX-E12 cells exposed to fermentation supernatants. Despite some donor-dependent differences, all three tested products showed beneficial modulatory effects on microbial activity represented by an increase in lactate and SCFA levels (acetate, butyrate and to a lesser extent also propionate), while decreasing proteolytic markers. Bifidogenic effect of XOS was consistent, while AOB supplementation appears to exert a specific impact on reducing F. nucleatum and increasing butyrate-producing B. wexlerae . Functional and compositional microbial changes were translated to an in vitro host response by increases of the intestinal barrier integrity by all the products and a decrease of the redox potential by AOB supplementation.

The prevalence of acute respiratory infections and their impact on quality of life underlies the need for efficacious solutions that are safe, sustainable and economically viable. Polysaccharides in several (traditional) plant extracts have been shown to be immunostimulatory, and some studies suggest beneficial effects against respiratory infections. The aim of this study was to (i) identify the active polysaccharide constituents from affordable and renewable crops (bell pepper and carrot) using activity-guided fractionation, (ii) evaluate in vitro effects on innate immune responses (phagocytosis and cytokine secretion), microbiota modulation and production of short chain fatty acids, followed by (iii) the evaluation of effects of a bell pepper extract enriched for the active component in a human proof of concept study. We identified rhamnogalacturonan-I (RG-I) as the nutricophore responsible for the immunostimulatory activity with substantial structural and functional equivalence between bell pepper (bp) and carrot (c). The in vitro studies showed that bpRG-I and cRG-I comprise similar immune- and microbiota modulatory potential and the human study demonstrated that bpRG-I was well tolerated and enhanced innate immune responsiveness in vivo. This is an important step towards testing the efficacy of RG-I from bpRG-I or cRG-I in an infection trial in humans.

Because of the recognized health benefits of breast milk, it is recommended as the sole nutrition source during the first 6 months of life. Among the bioactive components are human milk oligosaccharides (HMOs) that exert part of their activity via the gut microbiota. Here, we investigated the gut microbiota fermentation of HMO 2'fucosyllactose (2'-FL), using two in vitro models (48 h fecal incubations and the long-term mucosal simulator of the human intestinal microbial ecosystem [M-SHIME ® ]) with fecal samples from 3-month-old breastfed (BF) infants as well as 2-3 year old toddlers. The short-term model allowed the screening of five donors for each group and provided supportive data for the M-SHIME ® study. A key finding was the strong and immediate increase in the relative abundance of Bifidobacteriaceae following 2'-FL fermentation by both the BF infant and toddler microbiota in the M-SHIME ® . At the metabolic level, while decreasing branched-chain fatty acids, 2'-FL strongly increased acetate production together with increases in the health-related propionate and butyrate whilst gas production only mildly increased. Notably, consistently lower gas production was observed with 2'-FL fermentation as compared to lactose, suggesting that reduced discomfort during the dynamic microbiome establishment in early life may be an advantage along with the bifidogenic effect observed.

Background: This study evaluated the impact of Bifidobacterium animalis ssp. lactis CNCM I-3446, Bovine Milk-derived OligoSaccharides (BMOS) and their combination on infant gut microbiota in vitro. In addition, a novel strategy consisting of preculturing B. lactis with BMOS to further enhance their potential synbiotic effects was assessed., Method: Short-term fecal batch fermentations (48 h) were used to assess the microbial composition and activity modulated by BMOS alone, B. lactis grown on BMOS or dextrose alone, or their combinations on different three-month-old infant microbiota., Results: BMOS alone significantly induced acetate and lactate production (leading to pH decrease) and stimulated bifidobacterial growth in 10 donors. A further in-depth study on two different donors proved B. lactis ability to colonize the infant microbiota, regardless of the competitiveness of the environment. BMOS further enhanced this engraftment, suggesting a strong synbiotic effect. This was also observed at the microbiota activity level, especially in a donor containing low initial levels of bifidobacteria. In this donor, preculturing B. lactis with BMOS strengthened further the early modulation of microbiota activity observed after 6 h., Conclusion: This study demonstrated the strong synbiotic effect of BMOS and B. lactis on the infant gut microbiota, and suggests a strategy to improve its effectiveness in an otherwise low- Bifidobacterium microbiota.

Modulation of the gut microbiome as a means to improve human health has recently gained increasing interest. In this study, it was investigated whether cRG-I, a carrot-derived pectic polysaccharide, enriched in rhamnogalacturonan-I (RG-I) classifies as a potential prebiotic ingredient using novel in vitro models. First, digestion methods involving α-amylase/brush border enzymes demonstrated the non-digestibility of cRG-I by host-derived enzymes versus digestible (starch/maltose) and non-digestible controls (inulin). Then, a recently developed short-term (48 h) colonic incubation strategy was applied and revealed that cRG-I fermentation increased levels of health-promoting short-chain fatty acids (SCFA; mainly acetate and propionate) and lactate comparable but not identical to the reference prebiotic inulin. Upon upgrading this fermentation model by inclusion of a simulated mucosal environment while applying quantitative 16S-targeted Illumina sequencing, cRG-I was additionally shown to specifically stimulate operational taxonomic units (OTUs) related to health-associated species such as Bifidobacterium longum , Bifidobacterium adolescentis , Bacteroides dorei , Bacteroides ovatus , Roseburia hominis , Faecalibacterium prausnitzii , and Eubacterium hallii . Finally, in a novel model to assess host-microbe interactions (Caco-2/peripheral blood mononuclear cells (PBMC) co-culture) fermented cRG-I increased barrier integrity while decreasing markers for inflammation. In conclusion, by using novel in vitro models, cRG-I was identified as a promising prebiotic candidate to proceed to clinical studies.

: Background: Prebiotics used as a dietary supplement, stimulate health-related gut microbiota (e.g., bifidobacteria, lactobacilli, etc.). This study evaluated potential prebiotic effects of an artichoke aqueous dry extract (AADE) using in vitro gut model based on the Simulator of Human Intestinal Microbial Ecosystem (SHIME ® )., Methods: Short-term colonic fermentations (48 h) of AADE, fructo-oligosaccharides (FOS), and a blank were performed. Microbial metabolites were assessed at 0, 6, 24, and 48 h of colonic incubation via measuring pH, gas pressure, lactate, ammonium, and short-chain fatty acids (SCFAs) levels. Community composition was assessed via targeted qPCRs., Results: After 24 and 48 h of incubation, bifidobacteria levels increased 25-fold with AADE ( p < 0.05) and >100-fold with FOS ( p < 0.05) compared to blank. Lactobacillus spp. levels only tended to increase with AADE, whereas they increased 10-fold with FOS. At 6 h, pH decreased with AADE and FOS and remained stable until 48 h; however, gas pressure increased significantly till the end of study. Acetate, propionate, and total SCFA production increased significantly with both at all time-points. Lactate levels initially increased but branched SCFA and ammonium levels remained low till 48 h., Conclusion: AADE displayed prebiotic potential by exerting bifidogenic effects that stimulated production of health-related microbial metabolites, which is potentially due to inulin in AADE., Competing Interests: A.V., A.Z., and E.R. are employed by Euromed S.A. The study funders (Euromed S.A.) had no role in the design, collection, or analysis of the data.